fmicb-10-01169 May 28, 2019 Time: 14:28 # 1 ORIGINAL RESEARCH published: 28 May 2019 doi: 10.3389/fmicb.2019.01169 Metabarcoding Insights Into the Trophic Behavior and Identity of Intertidal Benthic Foraminifera Panagiota-Myrsini Chronopoulou1*, Iines Salonen1*, Clare Bird2, Gert-Jan Reichart3 and Karoliina A. Koho1 1 Aquatic Biogeochemistry Research Unit, Ecosystems and Environment Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland, 2 Biological and Environmental Sciences, University of Stirling, Stirling, United Kingdom, 3 Department of Ocean Systems, NIOZ-Royal Netherlands Institute for Sea Research and Utrecht University, Den Burg, Netherlands Foraminifera are ubiquitous marine protists with an important role in the benthic carbon cycle. However, morphological observations often fail to resolve their exact taxonomic placement and there is a lack of field studies on their particular trophic preferences. Here, we propose the application of metabarcoding as a tool for the elucidation of the in situ feeding behavior of benthic foraminifera, while also allowing the correct Edited by: taxonomic assignment of the feeder, using the V9 region of the 18S (small subunit; SSU) Ramiro Logares, Institute of Marine Sciences (ICM), rRNA gene. Living foraminiferal specimens were collected from two intertidal mudflats Spain of the Wadden Sea and DNA was extracted from foraminiferal individuals and from the Reviewed by: surrounding sediments. Molecular analysis allowed us to confirm that our foraminiferal Franck Lejzerowicz, University of California, San Diego, specimens belong to three genetic types: Ammonia sp. T6, Elphidium sp. S5 and United States Haynesina sp. S16. Foraminiferal intracellular eukaryote communities reflected to an Russel J. S. Orr, extent those of the surrounding sediments but at different relative abundances. Unlike University of Oslo, Norway sediment eukaryote communities, which were largely determined by the sampling site, *Correspondence: Panagiota-Myrsini Chronopoulou foraminiferal intracellular eukaryote communities were driven by foraminiferal species, [email protected] followed by sediment depth. Our data suggests that Ammonia sp. T6 can predate on Iines Salonen iines.salonen@helsinki.fi metazoan classes, whereas Elphidium sp. S5 and Haynesina sp. S16 are more likely to ingest diatoms. These observations, alongside the use of metabarcoding in similar Specialty section: ecological studies, significantly contribute to our overall understanding of the ecological This article was submitted to Aquatic Microbiology, roles of these protists in intertidal benthic environments and their position and function a section of the journal in the benthic food webs. Frontiers in Microbiology Keywords: metabarcoding, benthic foraminifera, trophic strategy, benthic food web, benthic microbial ecology, Received: 03 December 2018 molecular phylogeny Accepted: 07 May 2019 Published: 28 May 2019 Citation: INTRODUCTION Chronopoulou P-M, Salonen I, Bird C, Reichart G-J and Koho KA Benthic foraminifera are ubiquitous, single-celled protists. Due to their opportunistic character (2019) Metabarcoding Insights Into the Trophic Behavior and Identity (e.g., Moodley et al., 2000; Woulds et al., 2007), foraminifera can take advantage of their of Intertidal Benthic Foraminifera. environment very efficiently and they are able to thrive in a wide variety of marine environments. Front. Microbiol. 10:1169. Their ecology is complex, with some species harboring photosynthetically active symbionts or doi: 10.3389/fmicb.2019.01169 kleptoplasts (e.g., Hallock, 2000; LeKieffre et al., 2018; Schmidt et al., 2018) and other various Frontiers in Microbiology| www.frontiersin.org 1 May 2019| Volume 10| Article 1169 fmicb-10-01169 May 28, 2019 Time: 14:28 # 2 Chronopoulou et al. Trophic Behavior and Identity of Benthic Foraminifera endobionts (e.g., Bernhard, 2003; Tsuchiya et al., 2015; Bernhard is well-represented in public databases, and it captures a large et al., 2018), of which some may be used in direct carbon transfer eukaryotic diversity including that of protists (Amaral-Zettler to host foraminifera (Tsuchiya et al., 2018). Foraminifera are et al., 2009; Behnke et al., 2011; Pawlowski et al., 2011). However, generally considered as heterotrophic organisms with multiple this hypervariable region has not yet been considered for the feeding strategies. Of these, carnivory and predation are well- taxonomic placement of benthic foraminifera. documented among planktonic foraminifera (Boltovskoy and Here, for the first time, to the best of our knowledge, we Wright, 1976; Bé et al., 1977), however, for benthic foraminifera target the V9 hypervariable region of the 18S rRNA gene we rely only on experimental observations, which suggest that within benthic foraminiferal cells. In addition, the foraminiferal some species may pray on nematodes or other metazoans intracellular eukaryote communities are compared to those of (Suhr et al., 2008; Dupuy et al., 2010). Instead, a number their surrounding sediments to gain insights into the relative of experimental studies suggest that phototrophs provide an distribution of foraminiferal food sources in the sediment. important source of organic carbon and nutrients to benthic Moreover, the observed intracellular eukaryote diversity is linked foraminifera (Moodley et al., 2000; Nomaki et al., 2005, 2006; to external factors (e.g., site, habitat depth in sediment, and total Larkin et al., 2014; Jeffreys et al., 2015; LeKieffre et al., 2017). sedimentary organic carbon and nitrogen content), as parameters Generally, however, there is a distinct lack of in situ evidence like organic carbon availability and sediment depth have been of species-specific feeding modes and ecological relationships shown to be important in structuring the intertidal foraminifera among benthic foraminifera and sediment micro- and meiofauna community (e.g., Thibault De Chanvalon et al., 2015; Mojtahid due to the difficulties of studying these processes in nature. et al., 2016). The overall aim of this study is to identify species- Understanding species-specific feeding behaviors is crucial to specific trophic preferences of benthic foraminifera, and, in unraveling the adaptability strategies of benthic foraminifera in parallel to unravel their taxonomic identity. their habitats, understanding the benthic food webs structure and addressing implications for the global marine benthic biogeochemical cycles. MATERIALS AND METHODS Metabarcoding may provide new insights into life strategies and in situ feeding modes of foraminifera and allow the Site Description and Sampling identification of potential species-specific preferences. This Two intertidal mudflat localities (Supplementary Figure 1) approach has been successfully applied to investigate the were sampled in November 2015 at the Dutch Wadden microbiome and potential feeding preferences of marine Sea: Mokbaai (M) characterized by relatively sandy sediment eukaryotes, such as copepods (Ray et al., 2016) and nematodes with the presence of polychaete worm burrows (>10 cm (Schuelke et al., 2018). Recently, 16S rRNA metabarcoding was depth), and de Cocksdorp (C) characterized by non-burrowed also used to study the intracellular bacterial composition of clay/mud sediment. pelagic foraminifera to elucidate their ecological strategies (Bird One sediment core (10 cm internal diameter) per site was et al., 2017, 2018). Cloning and shallow Sanger sequencing sampled manually by pushing a core tube into the sediment have been recently used to demonstrate the multiple diatom during low tide and processed as described in Koho et al., associations within an individual benthic foraminifer, suggesting 2018; see detailed steps in Supplementary Figure 1C. In short, that the host can shuffle its symbionts in response to thermal three sub-cores (50 ml truncated syringes) were taken from stress (Schmidt et al., 2018). However, the application of the main core. Two of the sub-cores were transferred in a metabarcoding in benthic foraminifera is yet to be tested. nitrogen-filled glove bag and sliced with 1 cm intervals down to A good taxonomic resolution is essential in solving species- 10 cm depth. Porewater was removed, centrifuging the sediment, specific feeding preferences and potential niche and resource and the solid phase was frozen to –20◦C and transferred to partitioning among foraminiferal population. For planktonic the University of Helsinki, where it was freeze-dried. Then, foraminifera, cryptic species have been shown to display niche sedimentary organic carbon and total nitrogen was measured differentiation within the water column (Weiner et al., 2012) with a Leico TruSpec R Micro, following homogenization and as well as geographically on a spatial scale (Aurahs et al., decalcification (1 M HCl). The third sub-core was also sliced 2009). Metabarcoding allows not only the identification of prey at 1 cm intervals down to 10 cm sediment depth and used but also the cryptic diversity of the feeder that is not readily to obtain environmental DNA (eDNA; referred to as sediment distinguished morphologically (e.g., Miller et al., 1982; Pawlowski DNA) samples and foraminiferal specimens. Each sediment slice and Holzmann, 2008; Schweizer et al., 2011; Pillet et al., 2012; was subsampled (ca. 1–1.5 g sediment) with a sterile plastic Darling et al., 2016; Roberts et al., 2016; Lei et al., 2017). The 37f spatula, the subsample was immediately frozen in liquid nitrogen hypervariable